Project Summary/Abstract Diabetic kidney disease (DKD) affects up to 40% of patients with diabetes. Despite the increasing use of glucose- and blood pressure- lowering medication, the prevalence of DKD is on the rise. Injury and dysfunction of the specialized epithelial cells in the kidney glomeruli have been shown to be a fundamental component of DKD pathology. However, therapeutic discovery for DKD, and kidney disease in general, has lagged other areas due to the lack of assays that faithfully capture the complex pathophysiology of nephropathy. Organs-on-chips are quickly emerging as novel in-vitro platforms to model diseases and test potentially therapeutic compounds. As organs-on-chips are adopted for this purpose, there is a need for in-depth platform characterization and validated disease models. Therefore, the development of a DKD model in a glomerulus-on-chip will serve as a valuable research tool. In this proposal, we seek to engineer a physiomimetic model of the glomerulus, which can be utilized to study the pathophysiology of DKD. To achieve this goal, a thorough engineering methodology will be implemented to design and build an organ-on-chip platform to recapitulate the microenvironment of the glomerular filtration barrier. Based on the output of the engineering design, rapid prototyping equipment will be used to build a PDMS-free organ-on-chip for cell culture and custom fluid handling systems will be implemented to recapitulate physiologic pressures found in the glomerulus. The differentiation and culture of a conditionally immortalized human podocyte cell line through exposure to microenvironmental stresses, such as extracellular matrix stiffness, chemical signals, and pressure gradients, will be optimized on the platform. It is hypothesized that inclusion of these microenvironmental features will enhance the expression of podocyte specific genes and proteins required for maintaining the glomerular filtration barrier. Next, functionality of the engineered glomerulus- on-chip will be assessed via a filtration assay using clinically relevant proteins. Finally, DKD will be induced and characterized on the glomerulus-on-chip platform. Induction of the disease phenotype will be accomplished by exposing cells to sera isolated from patients with DKD. This approach is more advantageous than artificial inducers of injury, which decrease the likelihood that potentially therapeutic compounds will be beneficial in a clinical context. It is hypothesized that exposure to patient sera will result in both phenotypic and functional changes. Changes in filtration function will be assessed by the same filtration assay used to demonstrate normal filtration function. Changes in signaling between glomerular endothelial cells and podocytes will also be assessed. Ultimately, this proposal aims to develop a novel glomerulus-on-chip, which will be able to model the pathophysiology of DKD. Hopefully, this platform will assist in the development of new therapeutic compounds to treat DKD.